Interaction of the Intron-Encoded Mobility Endonuclease I-PpoI with its Target Site

Abstract

Endonucleases encoded by mobile group I introns are highly specific DNases that induce a double-strand break near the site to which the intron moves. I-PpoI from the acellular slime mold Physarum polycephalum mediates the mobility of intron 3 (Pp LSU 3) in the extrachromosomal nuclear ribosomal DNA of this organism. We showed previously that cleavage by I-PpoI creates a four-base staggered cut near the point of intron insertion. We have now characterized several further properties of the endonuclease. As determined by deletion analysis, the minimal target site recognized by I-PopI was a sequence of 13 to 15 bp spanning the cleavage site. The purified protein behaved as a globular dimer in sedimentation and gel filtration. In gel mobility shift assays in the presence of EDTA, I-PpoI formed a stable and specific complex with DNA, dissociating with a half-life of 45 min. By footprinting and interference assays with methidiumpropyl-EDTA-iron(II), I-PpoI contacted a 22- to 24-bp stretch of DNA. The endonuclease protected most of the purines found in both the major and minor grooves of the DNA helix from modification by dimethyl sulfate (DMS). However, the reactivity to DMS was enhanced at some purines, suggesting that binding leads to a conformational change in the DNA. The pattern of DMS protection differed fundamentally in the two partially symmetrical halves of the recognition sequence.

Profile of the DNA Recognition Site of the Archaeal Homing Endonuclease I-DmoI

“…4) are comparable with those reported earlier for the eukaryotic endonucleases I-PpoI and PI-SceI that cleave DNA by a similar mechanism to produce a 3′-OH 4 bp overhang, although only the latter carries the LAGLIDADG motifs (21,22). The former, I-PpoI, is also exceptional in that it binds as a dimer to an imperfect palindrome that is symmetrical with respect to the cleavage sites (23). I-DmoI and I-PpoI produce similar protection patterns when probed with DMS and they both produce the same hypersensitive site at A(+4) on the non-coding strand (23).…”

“…The former, I-PpoI, is also exceptional in that it binds as a dimer to an imperfect palindrome that is symmetrical with respect to the cleavage sites (23). I-DmoI and I-PpoI produce similar protection patterns when probed with DMS and they both produce the same hypersensitive site at A(+4) on the non-coding strand (23). PI-SceI-DNA complexes have not been probed with DMS but hydroxyl radical probing produced similar protection patterns on either side of, but not within, the cleavage region (24); these data were also reinforced by damage-selection experiments using methylation and ethylation reactions (17).…”

DNA binding and cleavage by the periplasmic nuclease Vvn: a novel structure with a known active site

“…A similar ‘ββα–metal’ topology has been revealed in the active site regions of several endonucleases (Friedhoff et al ., 1999b; Kuhlmann et al ., 1999; Miller et al ., 1999), including the homing endonuclease I‐ Ppo I (Flick et al ., 1998), Serratia nuclease (Miller et al ., 1994), phage T4 Endo VII (Raaijmakers et al ., 1999), and the H‐N‐H ColE7 (Ko et al ., 1999; Cheng et al ., 2002) and ColE9 (Kleanthous et al ., 1999). Among these DNases, I‐ Ppo I is the only site‐specific endonuclease recognizing and cleaving dsDNA with a specific sequence of 13–15 bp (Ellison, 1993). Serratia nuclease (Friedhoff et al ., 1996) and ColE7 (Ku et al ., 2002) are non‐specific endonucleases cleaving ds or ssDNA and RNA with little sequence preferences.…”

Elucidation of the catalytic mechanism of a single-metal dependent homing endonuclease using QM and QM/MM approaches: the case study of I-PpoI

“…1a-b). 38,39 Nevertheless, despite being one of the most well-characterized members of HE family through experimental biochemical and structural techniques, 20,21,39,[46][47][48][49][50][51][52][53][54][55][56][57][58][59][60] no consensus has been reached regarding the I-PpoI catalytic mechanism.…”